Osteoimmune Properties of Mesoporous Bioactive Nanospheres: A Study on T Helper Lymphocytes

Abstract

Bioactive mesoporous glass nanospheres (nanoMBGs) charged with antiosteoporotic drugs have great potential for the treatment of osteoporosis and fracture prevention. In this scenario, cells of the immune system are essential both in the development of disease and in their potential to stimulate therapeutic effects. In the present work, we hypothesize that nanoMBGs loaded with ipriflavone can exert a positive osteoimmune effect. With this objective, we assessed the effects of non-loaded and ipriflavone-loaded nanoparticles (nanoMBGs and nanoMBG-IPs, respectively) on CD4⁺ Th2 lymphocytes because this kind of cell is implicated in the inhibition of osseous loss by reducing the RANKL/OPG relationship through the secretion of cytokines. The results indicate that nanoMBGs enter efficiently in CD4⁺ Th2 lymphocytes, mainly through phagocytosis and clathrin-dependent mechanisms, without affecting the function of these T cells or inducing inflammatory mediators or oxidative stress, thus maintaining the reparative Th2 phenotype. Furthermore, the incorporation of the anti-osteoporotic drug ipriflavone reduces the potential unwanted inflammatory response by decreasing the presence of ROS and stimulating intracellular anti-inflammatory cytokine release like IL-4. These results evidenced that nanoMBG loaded with ipriflavone exerts a positive osteoimmune effect.

Keywords: ipriflavone; lymphocytes; mesoporous bioactive glass; nanospheres; osteoimmunology; osteoporosis.

Figure 1

Interaction between an APC MCH II and a CD4⁺ T lymphocyte: TCR-MHC II interaction and the subsequent CD28-CD80 interaction and effects of uncharged and ipriflavone-charged mesoporous bioactive glass nanoparticles, nanoMBGs and nanoMBG-IPs, on different cellular parameters on CD4⁺ Th2 lymphocyte.

Figure 2

Transmission electron images of (A) nanoMBGs and (B) nanoMBG-IPs.

Figure 3

Incorporation of nanoMBGs, cell size and complexity of CD4⁺ Th2 SR.D10 lymphocytes. Statistical significance: *** p < 0.005 dose effect; # p < 0.05, ## p < 0.01, ### p < 0.005 time effect.

Figure 4

nanoMBG incorporation by Th2 CD4⁺ SR.D10 lymphocytes and their viability in the presence of endocytic inhibitors. Statistical significance: *** p < 0.005.

Figure 5

Effect of nanoMBGs and nanoMBG-IPs on the cell cycle phases of CD4⁺ Th2 SR.D10 lymphocytes: SubG₁ (indicative of apoptosis), G₀/G₁ (quiescence/gap1), and S/G₂/M (synthesis/gap 2/mitosis). +IL = interleukin-enriched medium (25 pg/mL mrIL-1, 5 U/mL mrIL-2, and 10 U/mL mrIL-4), −IL = medium without interleukins. Statistical significance: * p < 0.05, *** p < 0.005 conditions vs. control; # p < 0.05 nanoMBGs vs. nanoMBG-IPs).

Figure 6

Effect of nanoMBGs and nanoMBG-IPs on the proliferation of Th2 CD4⁺ SR.D10 lymphocytes in basal and activated conditions with CD3, CD4, or both, after 24 h (A) and 48 h (B). No statistically significant differences were observed.

Figure 7

Effect of nanoMBGs and nanoMBG-IPs on the intracellular content of reactive oxygen species (ROS) (A) and viability (B) of Th2 CD4⁺ SR.D10 lymphocytes in basal and activated conditions with CD3, CD4, or both. Statistical significance: *** p < 0.005 Ct vs. Sph+F or Sph-F in each condition; ### p < 0.005 Sph-F vs. Sph+F in each condition; ++ p < 0.01, +++ p < 0.005 stimuli effect.

Figure 7

Effect of nanoMBGs and nanoMBG-IPs on the intracellular content of reactive oxygen species (ROS) (A) and viability (B) of Th2 CD4⁺ SR.D10 lymphocytes in basal and activated conditions with CD3, CD4, or both. Statistical significance: *** p < 0.005 Ct vs. Sph+F or Sph-F in each condition; ### p < 0.005 Sph-F vs. Sph+F in each condition; ++ p < 0.01, +++ p < 0.005 stimuli effect.

Figure 8

Effects of nanoMBG and nanoMBG-IP on the production of IL-4 (A) and IL-10 (B) by Th2 CD4⁺ SR.D10 lymphocytes under basal and stimulated conditions with CD3, CD4, or both. ND = Non-detected. Statistical significance: * p < 0.05, *** p < 0.005 conditions vs. control; # p < 0.05, ## p < 0.01, ### p < 0.005 stimulus effect.